Botulinum toxins (also known as botulinum neurotoxins) are neurotoxins produced by the gram-positive bacteria Clostridium botulinum. They act by causing the paralysis of the muscles through the inhibition of the release of acetylcholine in the presynaptic axon terminal of the neuromuscular junction (synaptic transmission), thus preventing nerve transmission and muscle contraction. The paralyzing effects of the muscles of the botulinum toxin have been used both for therapeutic purposes as well as for cosmetic effects. The controlled administration of the botulinum toxin has been used for the treatment of a wide range of conditions, disorders and diseases, such as disorders and diseases of the urinary bladder (EP 2273976 A2), premature ejaculation (US 2011/052636 A1), priapism (U.S. Pat. No. 6,776,991 B2), ulcers and gastroesophageal reflux (U.S. Pat. No. 7,238,357 B2), disorders and diseases associated with hyper- and hypothyroidism (U.S. Pat. No. 6,740,321 B2), primary hyperparathyroid disorders and diseases (U.S. Pat. No. 6,974,793 B2), perspiration and hyperhidrosis (U.S. Pat. No. 6,974,578 B2 and U.S. Pat. No. 6,683,049 B2), inflammatory eye disorders and diseases (U.S. Pat. No. 7,465,458 B2 and U.S. Pat. No. 7,220,422 B2), strabismus (U.S. Pat. No. 6,841,156), otic disorders and diseases (U.S. Pat. No. 6,265,379 B2 and U.S. Pat. No. 6,358,926 B2), excess cerumen secretion (US 2010/028385), neuropsychiatric disorders and diseases such as Alzheimer's, anxiety, schizophrenia, mania, depression (U.S. Pat. No. 7,811,587 B2), different compulsive disorders and diseases such as obsessions, compulsive skin picking, Tourette's syndrome, trichotillomania (U.S. Pat. No. 7,393,537 B2), cerebral paralysis (U.S. Pat. No. 6,939,852 B2), gonadotropin-related disorders and diseases (WO 02/074327), different cancers (U.S. Pat. No. 6,139,845 B2, U.S. Pat. No. 7,838,007 B2), neoplasms (U.S. Pat. No. 7,709,440 B2), different types of pain including headaches, migraines, fibromyalgia, arthritis or neuropathic pain among others (US 2010/266638, U.S. Pat. No. 7,811,586 B2, U.S. Pat. No. 7,704,524 B2, U.S. Pat. No. 7,704,511 B2, U.S. Pat. No. 7,468,189 B2, U.S. Pat. No. 7,255,866 B2, U.S. Pat. No. 7,091,176 B2, U.S. Pat. No. 6,887,476 B2, U.S. Pat. No. 6,869,610 B2, U.S. Pat. No. 6,838,434 B2, U.S. Pat. No. 6,641,820 B2, U.S. Pat. No. 6,623,742 B2, U.S. Pat. No. 6,565,870 B1, U.S. Pat. No. 6,500,436 B1, U.S. Pat. No. 6,458,365 B1, U.S. Pat. No. 6,423,319 B1, U.S. Pat. No. 6,113,915 A and U.S. Pat. No. 5,714,468 A), neurogenic inflammation (U.S. Pat. No. 6,063,768 B2), different disorders and diseases of the autonomic nervous system such as otitis and sinusoidal disorders (U.S. Pat. No. 5,766,605 A), disorders and diseases of the smooth muscle (U.S. Pat. No. 5,437,291 A), nerve impingements (US 2003/0224019), epilepsy (U.S. Pat. No. 7,357,934 B2), dystonia (U.S. Pat. No. 6,872,397 B2), trembling (U.S. Pat. No. 6,861,058 B2), Parkinson's disease (U.S. Pat. No. 6,620,415 B2), dizziness (U.S. Pat. No. 7,270,287 B2), osteoporosis (WO 2011/038015), different disorders and diseases of the skin such as calluses, warts, ulcers and lesions on the skin (U.S. Pat. No. 8,048,423 B2, US 2011/206731), psoriasis and dermatitis (U.S. Pat. No. 5,670,484 A), vascular hyperreactivity and rosacea (WO 2010/114828), acne (WO 03/011333), hair growth and maintenance (U.S. Pat. No. 6,299,893 B1), facial wrinkles (U.S. Pat. No. 7,255,865 B2), ptosis of the eyebrows and forehead (US 2011/280978) or drooping mouth corners (U.S. Pat. No. 6,358,917 B1) among others.
However, the toxicity inherent in botulinum toxin causes its administration, in a wide range of doses, to result in undesired secondary effects, such as immunogenic responses, cephalalgias, nausea, paralysis or muscle weakness, respiratory failure, and in more extreme cases even the death of the subject treated [FDA News, Feb. 8, 2008, “FDA Notifies Public of Adverse Reactions Linked to Botox Use”; Coté, T. R. et al. “Botulinum toxin type A injections: Adverse events reported to the US Food and Drug Administration in therapeutic and cosmetic cases” J. Amer. Acad. Derm. 2005, 53 (3), 407-415]. These severe secondary effects, together with the high cost of the treatment, seriously limits the application of botulinum toxin with therapeutic or cosmetic purposes, being relegated to chronic applications and/or diseases for which there is no suitable treatment. There is, therefore, a pressing need to develop molecules which imitate the paralyzing effects of botulinum toxins but which are equipped with much simpler and more stable molecular structures that do not induce immune reactions, and whose cost of production is affordable. Molecules of a peptide nature comply with these properties.
At a molecular level, botulinum toxins are proteases which degrade neuronal proteins that are involved in the exocytosis mechanism activated by the calcium ion [Schiavo G. et al. “Bases Moleculares del tétanos y del botulismo” Investigación y Ciencia 1996, 234, 46-55; Montecucco C. and Schiavo G. “Mechanism of action of tetanus and botulinum neurotoxins” Mol. Microbiol. 1994, 13, 1-8; Schiavo G. et al. “Tetanus and botulinum neurotoxins are zinc proteases specific for components of the neuroexocytosis apparatus” Ann. NY Acad. Sci. 1994, 710, 65-75]. For example, botulinum toxin A, the most commonly used in clinics to treat the symptomatology of spasmodic diseases and in cosmetics due to its applications in the elimination of facial wrinkles and facial asymmetry, breaks down the neuronal protein SNAP-25. This protein SNAP-25 plays a key role in neurosecretion since it is involved in the formation of a protein complex (known by the name of SNARE or fusion complex) which manages and controls the release of acetylcholine accumulated in vesicles. The nucleus of this fusion complex is comprised of the proteins SNAP-25 and syntaxin, located in the presynaptic plasma membrane, and the synaptobrevin protein of the VAMP family of proteins, located in the vesicular plasma membrane [Calakos N. and Scheller R. H. “Synaptic vesicle biogenesis, docking and fusion: a molecular description” Physiol. Rev. 1996, 76, 1-29; Sutton R. B. et al. “Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution” Nature 1998, 395, 347-353]. The principal function of the fusion complex is to bring the neurotransmitter (acetylcholine) loaded vesicle closer to and place it in contact with the presynaptic plasma membrane [Calakos N. and Scheller R. H. “Synaptic vesicle biogenesis, docking and fusion: a molecular description” Physiol. Rev. 1996, 76, 1-29; Sutton R. B. et al. “Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4Å resolution” Nature 1998, 395, 347-353]. In this way, in response to an increase in the concentration of calcium, the fusion of both plasma membranes will be favored, thus producing the release of the neurotransmitter. Therefore, this vesicle docking and fusion protein SNARE complex constitutes a key target for controlling neurosecretion. The truncation of any of the proteins which form the fusion complex prevents their assembling and, therefore, inhibits vesicle release and inhibits neuronal exocytosis.
It is known in the prior art that certain peptides derived from the protein sequences which form the SNARE complex are capable of inhibiting neuronal exocytosis, such as peptides derived from the amino and carboxy-terminal domains of the protein SNAP-25 [Apland J. P. et al. “Peptides that mimic the carboxy-terminal domain of SNAP-25 block acetylcholine release at an aplysia synapse” J. Appl. Toxicol. 1999, 19, Suppl. 1: S23-S26; Mehta P. P. et al. “SNAP-25 and synaptotagmin involvement in the final Ca2+-dependent triggering of neurotransmitter exocytosis” Proc. Natl. Acad. Sci. USA 1996, 93, 10471-10476; Ferrer-Montiel A. V. et al. “The 26-mer peptide released from cleavage by botulinum neurotoxin E inhibits vesicle docking” FEBS Lett. 1998, 435, 84-88; Gutierrez L. M. et al. “A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca2+-dependent exocytosis in chromaffin cells” FEBS Lett. 1995, 372, 39-43; Gutierrez L. M. et al. “A peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory vesicle docking in chromaffin cells” J. Biol. Chem. 1997, 272, 2634-2639; Blanes-Mira C et al. “Small peptides patterned after the N-terminus domain of SNAP-25 inhibit SNARE complex assembly and regulated exocytosis” J. Neurochem. 2004, 88, 124-135], the peptides derived from the sequence of syntaxin amino acids [Martin F. et al. “Inhibition of insulin release by synthetic peptides show that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca2+-but not for guanosine 5′-[gammathio]thriphosphate-induced secretion” Biochem. J. 1996, 320, 201-205], of the synaptobrevin [Cornille F. “Inhibition of neurotransmitter release by synthetic prolinerich peptides shows that the N-terminal domain of vesicle-associated membrane protein/synaptobrevin is critical for neuro-exocytosis” J. Biol. Chem. 1995, 270, 16826-16830], of the synaptotagmin [Mehta P. P. et al. “SNAP-25 and synaptotagmin involvement in the final Ca2+-dependent triggering of neurotransmitter exocytosis” Proc. Natl. Acad. Sci. USA 1996, 93, 10471-10476] and of the protein snapin [Ilardi J. M. et al. “Snapin: A SNARE associated protein implicated in synaptic transmission” Nat. Neurosci. 1999, 2, 119-124]. Similarly, synthetic peptides obtained by rational design or by searching synthetic libraries which are capable of inhibiting neuronal exocytosis by interfering in the formation of the SNARE complex have also been described [Blanes-Mira C. et al. “Identification of SNARE complex modulators that inhibit exocytosis form an α-helix constrained combinatorial library” Biochem J. 2003, 375, 159-166].
The industrial application of this type of compounds has been limited. The document EP 2318033 A2 describes the use of peptides derived from SNAP-25 for the treatment of pain and inflammation, and the document EP 1856139 A2 describes the use of peptides derived from SNAP-25 chemically modified to increase their bioavailability for the treatment of different diseases for which the treatment with botulinum toxin has shown effectiveness, among them the treatment of hyperhidrosis. Similarly, the cosmetic industry has made significant efforts to develop compounds which imitate the action of botulinum toxins with use in the treatment and prevention of the formation of expression wrinkles [Blanes-Mira C. et al. “A synthetic hexapeptide (Argireline®) with anti-wrinkle activity” Int. J. Cosmetic Sci. 2002, 24, 303-310]. In particular, peptides derived from the amino terminal fragment of the protein SNAP-25 which have anti-wrinkle effects are described in the documents EP 1180524 A1 and EP 2123673 A1, international application WO 97/34620 also describes peptides derived from the sequence of amino acids of the protein SNAP-25, in particular from its carboxy-terminal region, or from the synaptobrevin or the syntaxin capable of inhibiting neuronal exocytosis, and international application WO 2011/048443 describes peptides derived from the subunit c of the membrane component of V-ATPase capable of inhibiting neuronal exocytosis through its bonding to synaptobrevin and its potential application as anti-wrinkle treatment.
Thus, this invention provides an alternative to the existing needs and comprises the discovery of peptide sequences not derived from the protein SNAP-25 which are capable of inhibiting neuronal exocytosis.